Sandwich ELISA assay
Summary
TLDRThis video tutorial delves into the specifics of sandwich ELISA, a highly sensitive technique for detecting antigens in biological samples. The process involves using two distinct monoclonal antibodies to 'sandwich' the antigen, resulting in a more precise and sensitive detection method compared to direct or indirect ELISA. The tutorial explains the steps of the sandwich ELISA, from fixing the first antibody to the addition of the sample, washing, and the use of a secondary enzyme-linked antibody to produce a colorimetric reaction indicating the presence of the antigen. Advantages include higher sensitivity and the ability to use crude samples without purification, while the challenge lies in the need for a deep understanding of the antigen and the design of specific antibodies for accurate detection.
Takeaways
- 🧪 Sandwich ELISA is a specific branch of ELISA techniques that is more sensitive, being two to five times more sensitive than direct or indirect ELISA.
- 🔍 The purpose of ELISA is to detect antigens, which are indicative of infections or infective agents in the body.
- 📚 Antigens are fragments of bacterial cells or viral particles, and their detection can help identify specific diseases.
- 🤝 In Sandwich ELISA, the antigen is 'sandwiched' between two different antibodies, hence the name.
- 🧬 Two specific monoclonal antibodies are required for Sandwich ELISA, each binding to different epitopes on the same antigen.
- 🛠️ The ELISA process involves using microplates with wells where one antibody is fixed at the bottom, and the antigen is detected through a series of steps.
- 🚿 Washing is a crucial step in ELISA to remove unbound antigens or antibodies, ensuring accurate results.
- 🔬 The detection process uses a primary antibody to bind the antigen and a secondary antibody linked to an enzyme for signal detection.
- 🎨 The addition of a substrate results in a color change, indicating the presence of the antigen of interest in the sample.
- 🚀 Sandwich ELISA offers advantages like higher sensitivity and does not require sample purification, making it more efficient.
- ⚠️ The main disadvantage of Sandwich ELISA is the complexity in designing the experiment, requiring a good understanding of the antigen and the design of two specific antibodies.
Q & A
What is the main focus of the video tutorial?
-The video tutorial focuses on explaining the sandwich ELISA technique, a branch of the ELISA technique, which is more sensitive compared to direct or indirect ELISA.
What is the purpose of performing an ELISA process?
-The ELISA process is performed for the detection of antigens present in the body, which can indicate the presence of infections or infective agents, thus helping in diagnosing diseases.
Why is the technique called 'sandwich ELISA'?
-The technique is called 'sandwich ELISA' because the antigen is trapped between two antibodies, resembling a sandwich with something in the middle and two things on top.
How many antibodies are required in a sandwich ELISA technique?
-Two different specific antibodies are required in a sandwich ELISA technique, each binding to a different epitope of the same antigen.
What is the role of the first antibody in the sandwich ELISA technique?
-The first antibody, also known as the fixed antibody, is attached to the bottom of the well in the ELISA plate and is specific to bind with the antigen of interest.
What is the purpose of the wash step in sandwich ELISA?
-The wash step is crucial to remove any unbound antigens and antibodies, ensuring that only the bound antigens and antibodies contribute to the final result, thus avoiding false positives.
What is the function of the secondary antibody in sandwich ELISA?
-The secondary antibody in sandwich ELISA binds to the primary antibody that is attached to the antigen and is linked to an enzyme, which is necessary for the detection process by breaking down a substrate into a colored product.
Why is sandwich ELISA considered more sensitive than direct or indirect ELISA?
-Sandwich ELISA is more sensitive because it uses two antibodies to capture the antigen, which increases the signal and allows for detection of the antigen at lower concentrations.
What are the advantages of sandwich ELISA over other types of ELISA?
-Sandwich ELISA offers higher sensitivity, does not require sample purification, and is very specific for the target antigen, making it suitable for complex mixtures.
What is a potential disadvantage of sandwich ELISA compared to other types of ELISA?
-A potential disadvantage is the difficulty in designing the experiment, as it requires a good understanding of the antigen and the design of two antibodies specific to different epitopes of the antigen.
Outlines
🧪 Introduction to Sandwich ELISA Technique
This paragraph introduces the concept of Sandwich ELISA, a specific branch of the enzyme-linked immunosorbent assay (ELISA) technique. It emphasizes the sensitivity of Sandwich ELISA, which is two to five times more sensitive than direct or indirect ELISA methods. The paragraph explains that ELISA is used for detecting antigens, which are indicative of infections or foreign substances in the body. The process of Sandwich ELISA involves trapping an antigen between two antibodies, hence the name 'sandwich.' The explanation includes the preparation of monoclonal antibodies that bind to different epitopes of the antigen of interest, which is crucial for the specificity of the test. The paragraph also describes the initial steps of the ELISA process, including the use of microplates with wells that contain the fixed antibody.
🔍 Detailed Process of Sandwich ELISA
This paragraph delves into the step-by-step process of conducting a Sandwich ELISA. It begins with the addition of the patient's sample, which may or may not contain the specific antigen. The presence of the antigen leads to its binding with the fixed antibody in the well. Following this, a wash step is performed to remove any unbound antigens, ensuring specificity. The next step involves the addition of a tracking antibody, also known as the primary selective antibody, which binds to the antigen. This is followed by the addition of a secondary antibody linked to an enzyme, which binds to the primary antibody. The final step involves the addition of a substrate that, when broken down by the enzyme, produces a colored product. The development of color in the wells indicates the presence of the antigen. The paragraph highlights the importance of wash steps after each stage to prevent false positives and ensure accurate results.
📊 Advantages and Disadvantages of Sandwich ELISA
The final paragraph discusses the advantages and potential disadvantages of using the Sandwich ELISA technique. The main advantage is its high sensitivity, which makes it a preferred method for detecting even minute quantities of antigens. Additionally, it does not require sample purification, unlike direct or indirect ELISA, which saves time and cost. However, the technique demands a good understanding of the antigen and the design of a pair of antibodies that bind to different epitopes of the antigen. If the epitopes are too similar, the results may not be accurate. Once calibrated, the test can be reliably used for detecting the specific antigen of interest. The paragraph concludes with an invitation for viewers to like and subscribe to the channel for more educational content.
Mindmap
Keywords
💡Sandwich ELISA
💡Antigen
💡Antibody
💡Epitopes
💡Enzyme-Linked Antibody
💡Primary Antibody
💡Secondary Antibody
💡Wash Step
💡Sensitivity
💡Substrate
Highlights
Introduction to sandwich ELISA as a branch of ELISA technique with higher sensitivity compared to direct or indirect ELISA.
Explanation of the purpose of ELISA for detecting antigens which indicate infections or foreign substances in the body.
Description of antigens as fragments of bacterial or viral particles that can be tagged to specific diseases.
Overview of sandwich ELISA's process where an antigen is trapped between two antibodies, hence the 'sandwich' analogy.
Requirement for two different monoclonal antibodies specific to two different epitopes of the same antigen in sandwich ELISA.
Use of patient serum in ELISA due to its antigen content and the process of designing specific antibodies for antigen detection.
Explanation of the ELISA kit components, including micro plates with wells for antibody binding and antigen detection.
Process of fixing a specific antibody at the beginning of the ELISA process and its specificity to bind with only the target antigen.
Importance of the washing stage in ELISA to remove unbound antigens and ensure specificity of results.
Introduction of the tracking antibody in sandwich ELISA which identifies the antigen after the initial binding.
Role of the secondary antibody linked with an enzyme in the sandwich ELISA process for signal amplification.
Mechanism of color development in ELISA wells indicating the presence or absence of the target antigen.
Advantages of sandwich ELISA including higher sensitivity and no need for sample purification compared to other ELISA types.
Disadvantage of sandwich ELISA being the difficulty in designing the experiment due to the need for specific antibodies for different epitopes.
Practical application of sandwich ELISA in identifying specific antigens of interest in a complex mixture.
The calibration challenge in sandwich ELISA and the importance of proper antibody pairing for accurate results.
Encouragement for viewers to like and subscribe for more educational content on ELISA and related techniques.
Transcripts
back friends welcome to another video
session from shos biology and in this
video tutorial we'll be talking about
sandwich Eliza we have talked about
Eliza as a whole as a technique we've
already talked about it and it's my idea
to just go back there and understand
that process very well because in this
video I'm not going to talk very tiny
details of Eliza I just talk about a
branch of Eliza that is sandwich Eliza
and uh there are different branches also
called direct Eliza indirect Eliza and
stuff but there's a difference between
sandwich Eliza and direct indirect Eliza
sandwich Eliza is uh in this case much
more sensitive compared with direct or
indirect Eliza it's almost two to five
times more sensitive now first things
why we do Eliza Eliza process is done
due to the detection of antigens that
are present in our body you know
antigens are definite indications of
infection or infective agents in our
body antigen means the fragmentized
portion of bacterial cell or viral
particles in our body so if antigen is
present if we can detect a specific
antigen we can tag it with a specific
disease and we understand about that
disease for that person so we detect
diseases using the detection of antigens
using Eliza techniques or there are
other immunological techniques like Ria
and stuff in Sandwich Eliza the idea is
uh the name Sandwich came because the
process steps as we going to study
you'll see in this case antigen is
trapped between two antibodies so it's
something like
this if I draw it in very very basic
drawing for understanding this is the
antibody and it is actually stuck the
antigen is actually this is the antigen
it actually stuck
between two different antibodies so it
just looks like a sandwich something is
in the middle two things on the top so
that's why it's known as sandwich elizer
technique now any elizer technique the
idea is very simple the idea is this is
the antigen okay so patient patient
sample contains antigen mostly we use
patient serum because it contains
antigen so what we know we know specific
antibody that can bind with that antigen
let's say this is the antigen
Zed antigen Zed that we want to find in
patient's blood so we know about the
antigen Zed so we can design a specific
monoclonal antibody that can bind with
the antiz Z so we prepared that
monoclonal antibody this is the antibody
let's name it as antibody Zed which
binds with antigen Z so we can design it
in the lab and if we know about the
stuff that antigen Zed binds with
antibody Zed we can also design another
antibody that is uh that is that can be
a primary antibody or something that
antibody is also another monoc antibody
say Z2 let's say this is Z1 that will
also bind with antigen Zed so here what
we require we are trying to figure out
the presence of a specific antigen this
is antigen Z so in that case we require
two different specific antibody against
two different epitopes of that same
antigen because these two antibodies are
not binding to the same epitope because
if they bound that create problem we
want them to Bound in the opposite
region of the an so we need two separate
epitopes of the antigen where the
antibodies will bound so we need a
specific antigen which we need to figure
out which we need to detect along with
that we require two
antibody that are very specific towards
two different epitopes of that antigen
so once you know that then we ready to
go for the experiment so this is what we
need to know and this is what we create
in the lab and what we create is small
micro small plates those plates contain
Wells grooves if I draw draw this it
can't be drawn in the two dimensional
space like that but the idea is this is
small groups every group and group looks
something uh
like like this one after another there
will be groups group means antibodies
can be bound at the
bottom okay and you can add
antigen let's say here that the same
reaction if we go on this is the antigen
and again you can put some other
antibody there like that so this is how
the things will go on and we have a
series of that chamber that contains
small tiny groups filled with groups and
now what we do we fix a specific
antibody at the beginning this antibody
Z1 we fix it at the very beginning we
called him the fixed
antibody and we also know what what
antigen to detect so so the grooves only
contain the fixed antibody only that's
that's what it looks like the kit looks
looks like now so so say at the very
beginning it will look something like
this okay this is the beginning don't
forget about this Stu this is the
beginning antibody zed1 and we know this
antibody Z1 is specific to bind with
antigen Zed only it will not bind to any
other antigen so if now what we do we
add the
sample at the sample now if our sample
contains two there are two possibilities
it can contain the an
or may not contain the antigen if our
sample contains the antigen it is going
to attach and bind with antibody Zed one
because this is the antigen
Zed okay and then what we do we do a
wash up okay after applying the sample
we do a wash up why wash up is required
wash up is very very important stage
because this washing stage will help us
to remove any Unbound antigens okay and
because the bound antigens will be fixed
with the antibody because antibod is
anchored properly into the slide well or
the slide
basement so wash we wash them off now
let's say antigen is present so rest
Unbound antigens will be washed away now
the scenario is like this then what we
add we add another antibody remember I
told you we require two antibodies we
attach one antibody at the kit and also
we have another antibody in the solution
this antibody we have in the solution
solution and that solution also come
from the
kit okay this antibody solution and this
antibod is known as the tracking
antibody it's also known as the the uh
primary selective select selectable
antibody which selects or identifies the
antigen okay or tracking antibody
whatever you say this is also primary
antibody because you know primary
antibod is work is just to attach to the
antigen that's it primary anti an body
is not going to give you any expression
okay for the expression we need
secondary antibody so this is the thing
this sandwich is prepared then we have
the second solution that also comes from
the kit and that second solution
contains another antibody that is anti-
primary antibody that means that
antibody can interact with this previous
antibody and that antibody is secondary
because that is attached to enzyme
sometimes we can use a primary antibody
which is directly attached to the enzyme
but that is costlier to produce and the
specificity cannot maintain all the time
for that reason we use two different
antibodies for the detection this is
only Target and then we have the
solution two it contains secondary
antibody it's also enzyme linked
antibody okay so this secondary antibody
will come and join this whole process so
let me draw it here here so here we go
the whole process that we know of
antigen
attached the primary
antibody also attached let draw it small
way and now the secondary
antibody and secondary antibodies linked
with
enzyme the enzyme could be different H
rce peroxides or hrp is used generally
as enzymes here now this is the
secondary antibody this is the primary
antibody now this secondary antibody
once bind to this primary antibody with
the enzyme complex then we add some
substrate and they break this substrate
this enzyme will break this substrate
down into a product okay and that
product is
colorful and we get the color so in the
wells if you if you just just watch this
process
now you see development of colors in the
solution and development of colors will
indicate the samples that you use
whether they contains the antigen of
your interest or not okay so ultimately
but the idea or the mechanism behind
this is this that we talked about every
time we do each of the stages we do a
washer middle there's a wash there a
washing stage every time we doing it
because we don't want to keep any
Unbound antigens or antibodies in the
mixture because if antigen antibody even
it's floating in the mixture still it
going it's going to give us some color
we don't want to do that that will be
false negative we only want the fixed
antigens to give us the result only for
that we need wash up stages conut every
after every this stage okay and it will
produce the colored product we get the
idea that is known as sandwich Eliza
because antigen is getting sandwiched
between two antibodies
for the detection of the antigen now if
we talk about the advantages of Sandwich
Eliza over other types of Eliza sandwich
Eliza is much more sensitive compared
with direct or indirect Eliza two to
five times more
sensitive and another important thing
about the sandwich Eliza is that in this
case of Sandwich
Eliza that does not require this whole
process do not require uh in the
purification of the sample that we take
while in direct or indirect elizer we
need to go for some purification of the
sample or Preparation of the sample
phase that will take time as well as
some expense but in this case we don't
need that we just take the crude sample
extract and we use it why we can use
that why we cannot do that for direct or
indirect Eliza because as we are
separating as we are finding the antigen
based on two different antibody ENT
trapment it is very very specific so
even if you have a a complex mixture
with so many different things so many
antigens different types of antigens
with different epitopes but still this
process can be very handy to find you
the specific Target antigen of your
interest but if you use it for any other
type of Eliza it might not so that's the
very important advantage of Sandwich
Eliza that we know of okay but the
disadvantage is the disadvantage is at
the beginning of Designing this
experiment you need to know
you need to have a very good
understanding of the antigen that we
need to separate and also according to
that antigen you need to design a pair
of antibody that will bind to two
different epitopes of that antigen
because if the epitope ranges are same
or very close then it might not end up
with good results so the calibration of
this whole experiment is difficult
except for that once you calibrate the
stuff for a specific antigen you can run
that antigen as many times or samples as
many times as you want to to identify
your antigen of
Interest so that's all about sandwich
Eliza If you like this video please hit
the like button and definitely subscribe
to my channel to get more and more
videos like that the subscription link
is given here as well as in the bottom
so please do subscribe and keep watching
thank
you
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